Summary: Macrophages (MO) are the first cells infected with HIV-1 in vivo and act as a virus reservoir. Cytokines are potent modulators of HIV, which enhance and inhibit virus replication. We have conducted studies to identify cytokines or antagonists (anti-cytokine antibodies or soluble cytokine receptors) capable of inhibiting HIV replication in human MO or T cells. Subsequent to showing that HIV-infected MO produce high levels of M-CSF and MIP-1a, which enable an HIV/MO reservoir to be established, we found that antagonists to M-CSF inhibit HIV replication and reduce production of MIP-1a when added to infected MO in vitro. These M-CSF antagonists may prove beneficial as therapies for inhibiting HIV replication in vivo in MO by blocking production of virus, reducing chemokine recruitment of HIV susceptible cells, and preventing establishment/maintenance of HIV-infected MO reservoirs in vivo (JI 2000). The lymphokine IL-2, used for therapeutic restoration of CD4+ T cells in AIDS patients, has been reported to increase M-CSF in human monocytes and cause a transient burst of HIV mRNA in plasma after administration. We asked if MO were the source of released HIV and found that exposure of MO to IL-2 prior to HIV infection in vitro leads to a dramatic decrease in virus replication, which correlates with an IL-2-induced decrease of CD4 and CCR5 expression. Production of M-CSF was not enhanced, suggesting that IL-2 may be beneficial not only in restoring T cell function in AIDS patients, but also in preventing MO infection with HIV (AIDS 1998). Interferon-alpha (IFN-a) has potent anti-HIV activity in vitro but clinical utility only in AIDS patients with high levels of CD4+ T cells. IFN-a species vary in their ability to inhibit HIV replication in vitro, which may correlate with the varying effects in vivo. We have found that IFN-a components and molecular hybrid molecules are effective at inhibiting HIV-1 replication in MO, but show considerable variation in human T cells. This appears to correlate with observed differences in the ability of some hybrids to inhibit monocyte vs. T cell tropic strains of HIV-1. Ongoing studies will determine whether: 1) IFN-a recombinant hybrid species modulate CD4, CCR5 or CXCR4 expression in MO and T cells; 2) anti-viral activity correlates with anti-proliferative activity in primary T cells; and 3) whether an IFN-a species with low anti-proliferative and high anti-viral activity on both MO and T cells can be identified which may cause fewer toxic side effects when used clinically. NK cells are the first line of defense against virus-infected cells and produce cytokines that are biologically active on MO. We studied the ability of NK cells to produce cytokines capable of preventing HIV replication in MO. We found that NK cells produce a novel factor which prevents HIV-1 replication following virus entry in MO, but not T cells. Partial purification indicates that the inhibitory factor(s) is approximately 10 kD with a pI of 8-10. The small size and basic pI are characteristic of a chemokine, but the ability of this factor to inhibit replication of HIV, and not virus entry, indicate that it is not a chemokine blocking via binding to CCR5. Consistent with this, we find that antibodies to beta chemokines are unable to reverse the inhibition when the NK cell-derived factor is added to MO after HIV adsorption. However, these antibodies can reverse inhibitory activity that occurs when this factor is added during virus adsorption. Our data suggest that this NK cell factor is distinct from beta chemokines that inhibit HIV entry and that NK cells may play a significant role in the regulation of HIV-1 expression in human MO.